1. Field of the Invention
The present invention relates to systems and methods for fluorescence detection.
More specifically, the present invention relates to fluorescence detection assemblies for analysis of fluorescent signals from target substances.
2. Background
Flow cytometry is commonly used to differentiate various types of cells and other “formed bodies” comprising a biological fluid, e.g., whole blood. Conventional flow cytometers commonly comprise an optically-transparent flow cell, usually made of quartz, having a central channel through which a stream of cells to be individually identified is made to flow. Movement of the cell stream through the flow cell channel can be hydrodynamically entrained to the central longitudinal axis of the flow cell channel by a cell-free sheath liquid that concentrically surrounds the cell stream and flows along with the cell stream as it passes through the flow cell channel. As each cell passes through a cell-interrogation zone of the flow cell channel, it is irradiated with a focused beam of radiation (as commonly provided by a laser source). Upon impinging upon each cell, the laser beam is scattered in a pattern characteristic of the morphology, density, refractive index and size of the cell. Further, the spectral characteristics of the laser beam may act to excite certain fluorochromes associated with selected cells, as may be the case when a cell's DNA has been previously stained with such fluorochromes, or when a fluorochrome molecule has been previously conjugated with a selected type of cell, either directly or via an intermediate bead or the like. Photodetectors strategically positioned about the optical flow cell serve to convert the light scattered by each cell and the fluorescence emitted by the excited fluorochromes to electrical signals which, when suitably processed, serve to identify the irradiated cell. A conventional light scatter and fluorescence-sensing flow cytometer of the type noted above is disclosed in U.S. Pat. No. 7,392,908 to Frazier, the disclosure of which is incorporated herein by reference in its entirety.
As an alternative to positioning photodetectors directly about the optical flow cell, a light collector can be used to gather fluorescent light emitted by the excited fluorochromes. The light collector, which can be a group of lens elements, images the emitted light to a plurality of optical fibers. Each optical fiber transmits light to an array of photodetectors, which in turn convert the fluorescence emitted by the excited fluorochromes to electrical signals for analysis.
Various types of photodetector arrays have been used to separate light into discrete wavelengths to aid in fluorescence analysis. One such detector array is disclosed by “Improved Multilaser/Multiparameter Flow Cytometer for Analysis and Sorting of Cells and Particles,” John A. Steinkamp et al., Review of Scientific Instruments, Vol. 62, No. 11, pp. 2751-2764, November 1992 (“the Steinkamp publication”). The Steinkamp publication describes a detector configuration that includes four dichroic filters to separate light into discrete wavelengths. The dichroic filters are arranged in one line, and each filter except the last in line reflects a certain band of light to an associated detector and transmits the remaining bands to the next detector. The last filter reflects one band of light to a detector and transmits the remaining wavelengths of light to a final detector.
A second type of detector array configuration is disclosed by U.S. Pat. No. 4,244,045 to Nosu et al. (“the Nosu patent”). Optical fibers introduce a beam of light into the demultiplexer, which is designed to separate light into multiple wavelengths. The demultiplexer includes six optical filters, each of which transmits certain wavelengths therethrough and reflects light waves with wavelengths sufficiently different from the transmitted wavelengths. Three optical filters are arranged on each side of an avenue. A light beam is introduced into the array via an optical fiber and a collimating rod lens positioned in parallel to the avenue at an angle of incidence of 15 degrees. The first optical filter, on the opposite side of the avenue from the collimating lens, receives light directly from the collimating lens. The next four optical filters receive light from a reflected beam coming from another of the optical filters located on the opposite side of the avenue.
Detector assemblies such as that described by the Steinkamp publication and the
Nosu patent are generally prefabricated blocks. As a result, multiple detector assemblies must be used if it is desired to analyze multiple light beams, for example, light beams of different color spectrums. Another problem with known detector assemblies and demultiplexers is that the blocks must be prefabricated based on the number of wavelengths of light to be separated, i.e., each block is manufactured with space for a predetermined number of dichroic filters and detectors. As a result, these prefabricated detector assemblies are not customizable to accommodate multiple light beams, each of which may require separation of a different amount of wavelengths.
The present invention provides detector arrays that can be customized with regard to the number of optical inputs that can be analyzed in order to accommodate changing numbers of optical inputs. Such detector arrays can be adjusted such that the number of dichroic filters and detectors associated with each optical input can be adjusted in order to separate a light beam into more or fewer wavelengths without requiring multiple detector assemblies.